Project Summary (Abstract): Type I Diabetes (T1D) is an early onset autoimmune disease that culminates in the targeted destruction of insulin producing ? cells found within the pancreatic islets of Langerhans. The top two genetic susceptibility determinants associated with development of Type 1 Diabetes are related to antigen presentation and antigen availability (HLA and INS-VNTR). The identification of these and other susceptibility alleles highlight a potential deficiency in central and peripheral tolerance to islets antigens. Therefore, studies focused on the fundamental aspects of autoimmune T cell selection are crucial in identifying the key mechanisms that lead to the loss of central tolerance. It is still unclear how the intracellular signaling and transcriptional profiles differ between T cells selected on naturally expressed high affinity peptides (potential autoimmune antigens) versus low affinity peptides (normal positive selection) during thymic development. The mechanisms behind loss of self-tolerance appear to multifaceted and poorly understood. It is now evident that in addition to susceptibility alleles, autoimmune diabetes is also associated with post-translationally modified epitopes that serve as neo-antigens and aide in loss of self-tolerance. Pathogenic T cells that are specific for neo-antigens uniquely expressed in the pancreas may escape thymic selection by a process known as ignorance. The Non-Obese Diabetic mouse model offers an in vivo system to dissect the mechanisms of autoreactive T cell development in the thymus that is not feasible in humans. Unlike systems build on model antigens, the NOD mouse offers a genetically susceptible background where key target antigens undergo post-translational modification leading to spontaneous diabetes development. Therefore, it is of high importance to study selection of autoimmune TCRs that have spontaneously escaped negative selection in NOD mice. A limitation to such approach is the limited number of available autoantigen-specific TCR transgenic NOD mice. This application capitalizes on our strengths and experience in generating insulin specific TCR retrogenic mice as well as our in-depth knowledge of thymocyte development and TCR signaling. The scope of this application will therefore be limited to, and focused on, dissecting specific signaling mechanisms that allow thymic selection of autoimmune insulin and chromogranin reactive Class-II restricted TCRs. In Aim 1 of the proposed work, we will use the InsB:9-23 TCR contact mutant, p16A, to determine the cellular mechanisms for positive and negative selection of high and low insulin reactive thymocytes. In Aim 2, we will determine the impact of early thymic antigen exposure in neonatal mice on life-long tolerance to islet antigens. Information gathered from these studies may have a significant impact on our understanding of how autoreactive T cells escape deletion and how antigen specific Treg development occurs, leading to novel approaches to establish tolerance to islet antigens.